Comprehensive identification of SUMO2/3 targets and their dynamics during mitosis.

The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Abstract

During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.

A) Schematic outline of the protocol for growth and synchronization of cells in roller bottles. B) HeLa FRT TRex SUMO2 and HeLa FRT TRex SUMO2ΔGG cells were arrested in S phase by thymidine or synchronized in mitosis with thymidine and taxol, followed by provoking mitotic exit by the addition of ZM447439 for the indicated times. Cell lysates were analyzed by western blotting using antibodies against SUMO2/3, Cyclin B1 and Aurora A as markers for mitotic progression, and Vinculin. (★) indicates un-conjugated SUMO2 fusion proteins, (⧫) indicates endogenous SUMO2/3. C) Schematic outline of the quantitative proteomics strategy. Cells are grown in large-scale and synchronized in roller bottles as shown in A. SUMO2ΔGG cells were isotopically labelled with light “L”/R0K0 amino acids and arrested in prometaphase (grey), cells expressing SUMO2 were labelled with medium “M”/R6K4 amino acids and represented the mitotic exit stage one hour after ZM447439 addition (green), and cells expressing SUMO2 were labelled with heavy “H”/R10K8 amino acids and arrested in prometaphase (red). Equal amounts of cell lysate from the labelled populations were mixed and SUMO2 conjugated proteins were purified as described in . The SUMO2 conjugate enriched sample was separated by SDS-PAGE, digested with trypsin and analyzed by mass spectrometry (MS). Lysates from the different experimental conditions were analyzed by western blotting antibodies against FLAG, Cyclin B1, Aurora A and Vinculin to confirm conjugation state and mitotic stage. (★) indicates un-conjugated SUMO2 fusion proteins. D) Scatter plot of the entire data set from screen I and screen II. The plot is showing the value of log2(M/L) and log2(H/L) SILAC ratios that are used to identify SUMOylation targets. The red dashed line at log2(M/L) = 1 and log2(H/L) = 1 represents the cut-off ratio of ≥2 for the respective SILAC pairs. Each point represents a single identified protein, proteins identified in screen I are illustrated in blue and proteins identified in screen II are in purple. Identified proteins that are classified as SUMOylation hits are above the dashed cut-off line and are darker colored, whereas identified proteins classified as background is below the cut-off and lighter. E) Diagram showing the number of identified targets (hits) of SUMO2/3 modification in screen I (blue), screen II (purple) and the overlap (dark blue) of hits that are identified in both. F) Scatter plot with the correlation between the screen I and screen II log2(H/M) ratios of identified SUMO2/3 target proteins. Each point represents a SUMO2/3 target. The pearson correlation, R, is shown.

Graph showing the individual modification dynamic, log2(H/M), of the 445 SUMO2/3 targets identified in screen II (purple). The dashed lines represent the |log2(H/M)|≥1 cut-off for modification dynamics. Below the green line at log2(H/M) = −1, targets are SUMOylated more in mitotic exit, and above the red line at log2(H/M) = 1, targets are SUMOylated in prometaphase arrest and deSUMOylated upon mitotic exit. Targets between the two lines are modified by SUMO at a constant level through mitosis or are showing dynamic to a lesser extent. The 10 most dynamic targets that are modified upon mitotic exit and the 5 identified targets that preferably are SUMOylated in prometaphase are highlighted below the graph with their respective gene names. Names of identified SUMO pathway components (in bold) and known targets of SUMOylation in mitosis are shown, and arrows indicate their respective ratios.

A) Identification of RhoGDIα as a SUMO2/3 target. SILAC ratios and MS data from screen I and II. B) Schematic representation of RhoGDIα with the amino acid sequence around lysine residues K138 and K141. The consensus modification motifs are indicated and the lysines are highlighted in red. C) Purification of Venus-RhoGDIα or Venus-RhoGDIα K138R/K141R from taxol arrested cells using GFP-trap beads. SUMO conjugation pathway components, Ubc9 and PIAS1-4, were depleted by RNAi, expression of Venus-RhoGDIα fusion proteins in the generated stable HeLa FRT Trex cell lines was induced with doxycycline and ZM447439 was added for one hour as indicated. The SUMOylation states of purified RhoGDIα and RhoGDIα K138R/K141R from the different experimental conditions were analyzed by western blotting with antibodies specific for SUMO2/3 and RhoGDIα. D) Parental HeLa FRT and HeLa FRT TRex SUMO2 cells were arrested in S phase by thymidine or synchronized in mitosis with thymidine and taxol, followed by mitotic checkpoint override and progression by the addition of ZM447439 for the indicated times. Cell lysates were analyzed by western blotting using antibodies against Cyclin B1, RhoGDIα, RhoA and α-tubulin, and shows that RhoGDIα itself is stable in mitosis. E) Using 3 different siRNA oligos for each target, normal HeLa cells were depleted for RhoGDIα or RhoGDIβ using RNAi. Lysates were analyzed for depletion efficiency and RhoA stabilization by western blot with RhoGDIα and RhoA specific antibodies. F) Stable HeLa FRT TRex FLAG-RhoGDIα or FLAG-RhoGDIα K138R/K141R cells were depleted for endogenous RhoGDIα and arrested in mitosis with taxol. Rescue with exogenous RhoGDIα fusion proteins were titrated in with increasing concentrations of doxycycline (ng/ml) as indicated. Lysates were analyzed for depletion efficiency, expression level of exogenous RhoGDIα and RhoA stabilization by western blot with RhoGDIα and RhoA specific antibodies. G) Representative still images from time-lapse movies of stable HeLa cell lines expressing Venus-RhoGDIα and Venus-RhoGDIα K138R/K141R as they progress through an unperturbed mitosis. The DIC and Venus channels are shown and the time of nuclear envelope breakdown (NEBD), metaphase and anaphase is indicated. H) As F) but using Venus-RhoGDIα.